Collagen has a triple-helical structure in which three polypeptide strands are wound around each other and a plurality of collagen molecules of about 300 nm in length are associated and staggered from each other by 67 nm to form a long collagen fiber. Collagen is a principal protein which constitutes skins, tendons, bones and the like of, for example, fish, pigs and cattle, and the majority of collagen is found in the form of insoluble fiber in vivo.
Although collagen fiber is insoluble, it has advantages, for example, in that it is a biological component and thus highly safe and that, because of the high homology among animals, it is not likely to cause an immunoreaction. Therefore, application of collagen has been investigated in the fields of food products, medicines and cosmetics, and there have been developed a variety of solubilization methods and application methods for solubilized collagen. Examples of solubilized collagen include, in addition to those obtained by extracting a trace amount of a soluble collagen contained in a material such as an animal skin or bone with a dilute acid, collagen solubilized by an addition of an enzyme such as protease (Patent Literature 1) and collagen solubilized by an addition of an alkali (Patent Literature 2).
Meanwhile, since water-soluble collagen has a low thermal denaturation temperature when it is in a solubilized state having a large water content and being transparent, there is also disclosed collagen having an improved thermal denaturation temperature (Patent Literature 3). Water-insoluble associations of collagen having an isoelectric point in the pH range of cosmetic formulations had been considered unsuitable as cosmetic materials due to several reasons such as their opaque white color and heterogeneity; however, in Patent Literature 3, it was discovered that a collagen association obtained by adjusting an aqueous solution of collagen having an isoelectric point at a pH of 5.5 to 8.5 to have a pH in the vicinity of the isoelectric point has a notably high thermal denaturation temperature and such association was used as a cosmetic material. In Examples of Patent Literature 3, a collagen solution having an isoelectric point at about pH 7.0 was salt-precipitated by adding thereto sodium chloride to a final concentration of 5% by mass. The thus obtained collagen precipitates were dissolved with hydrochloric acid and the resulting solution was then neutralized with sodium hydroxide to yield a dispersion of collagen associations. An undenatured collagen molecule is rod-shaped and has a unique triple-helix structure; however, since an aqueous collagen solution thereof has a low heat resistance, the helical structure of the collagen molecule is easily disrupted by a heat treatment. In the above-described Patent Literature 3, using undenatured collagen molecules that were obtained, the adhesion thereto of human epidermal cell was evaluated. It was shown that, in the case of gelatin, the human epidermal cell did not at all exhibit adhesion after 30 minutes of culturing and a very slight adhesion was observed after 3 hours of culturing; however, in the cases where undenatured collagen molecules were used, notable adhesion was observed after 30 minutes of culturing.
An undenatured collagen solution may be denatured even at normal temperature; therefore, storage thereof requires temperature control by refrigeration or the like. In view of such storage stability of collagen, there is disclosed a method of using a dry collagen (Patent Literature 4). In this method, a collagen solution is injected via a nozzle into a volatile hydrophilic organic solvent medium to yield a fibrous or membranous product and the thus obtained fibrous product or the like is then dried and shredded or pulverized to produce a granular or powdery collagen dry product. It is described that the collagen solution to be injected into the hydrophilic organic solvent medium preferably has a collagen concentration of 3 to 10% by mass and that the injection rate from the nozzle is preferably 1 to 30 m/min.
Furthermore, there is also disclosed a dried product of a collagen derivative originated from fish skin (Patent Literature 5). The invention of Patent Literature 5 is characterized in that a treatment with an organic solvent and a centrifugation treatment are performed in combination in order to reduce the odor of fish-originated collagen and that a collagen derivative such as an acylated collagen is prepared as collagen. In Example 2, after subjecting fish skin to an organic solvent treatment and a centrifugation treatment, the resulting collagen was solubilized with acetic acid and salt precipitation was performed to yield collagen precipitates, which were then freeze-dried to obtain dry collagen. Further, in Example 4, after subjecting fish skin to an organic solvent treatment and a centrifugation treatment and adding sodium hydroxide to the resulting precipitates, the resulting solution was stirred overnight and then centrifuged. Thereafter, an aqueous citric acid solution was added to the precipitates to extract collagen, which was then precipitated with an addition of sodium citrate, and the solution was adjusted to have a pH of 10 by adding a sodium hydroxide solution to the thus obtained collagen precipitates. Then, succinic anhydride was allowed to react with the collagen precipitates to obtain a succinylated collagen, which was subsequently precipitated with hydrochloric acid. Finally, the resulting precipitates were freeze-dried to obtain dry collagen product.
Moreover, collagen has different isoelectric point depending on the treatment method and a collagen solubilized under acidic conditions has an isoelectric point at a pH of 7 to 9.5; therefore, such a collagen has poor solubility at a pH of 5 or higher and forms precipitates and aggregates. In view of this point, there is also disclosed a method in which the water-solubility of collagen is improved by esterification (Patent Literature 6). In Patent Literature 6, an esterification reaction of collagen is performed in advance in an animal tissue condition and then extraction operations of the thus esterified collagen are performed. It is described that an esterified collagen can be thereby produced inexpensively with simple steps.